This project is concerned with the study of thionucleotides in tRNA. The best known function of thionucleotides involves the 2-thiouridine derivatives found in the anticodon of a number of prokaryotic and eukaryotic tRNAs. Such thionucleotides have been shown to be essential to precise codon recognition. At present, three mammalian tRNAs are known to contain 2-thiouridine derivatives in the anticodon; these include one isoaccepting species each of tRNAGlu, tRNAGln, and tRNALys. In previous studies, we isolated a tRNA sulfurtransferase system from the cytosol of the liver of Buffalo rats. This enzyme is capable of thiolating tRNA in vitro by catalyzing the transfer of labeled sulfur from (35S)Beta-mercaptopyruvate to tRNA, forming thionucleotides. The activity of this enzyme is markedly decreased in Morris hepatomas compared to normal rat liver and is due to the presence of an inhibitor in the tumor cytosol which suppresses the transsulfuration reaction. To determine whether this inhibitor of the in vitro transsulfuration reaction has a significance in vivo in terms of suppressing tRNA thiolation and causing derangements of tRNA function, we propose the following studies: 1) The three species of tRNAs known to contain a 2-thiouridine derivative in the anticodon will be isolated from normal rat liver and sequenced by post-labeling methods with 32P. 2) The three corresponding tRNAs from the fast-growing Morris hepatoma 9A will be isolated and sequenced similarly. 3) If the tumor tRNAs show deficient thiolation in the anticodon, their coding properties will be studied and compared with those of the corresponding normal liver tRNAs. 4) The pair of normal and tumor tRNAs showing the most divergence in primary structures will be examined by high-resolution NMR spectroscopy to determine how abnormalities in the primary structure of the tumor tRNA can affect its three-dimensional structure and in turn its biological function.